Inhibitors of the G protein G?s which drives pancreatic tumorigenesis Project Summary / Abstract The GNAS gene encodes the G?s stimulatory subunit of heterotrimeric G proteins, which mediate G-protein- coupled receptor (GPCR) signaling, a central mechanism by which cells sense and respond to extracellular stimuli. Multiple human cancer types exhibit recurrent gain-of-function mutations in the pathway, most frequently targeting GNAS. The most lethal tumor type where GNAS is frequently mutated is the intraductal papillary mucinous neoplasms (IPMN), a precursor of invasive pancreatic cancer. Recent mouse modelling from Bardeesy and coworkers has shown that pancreatic IPMN tumors which contain three coincident genetic lesions, K-Ras (G12D), G?s (R201C), and p53 -/-, are suppressed when G?s (R201C) is silenced, providing strong genetic validation for targeting this mutant protein. For over 30 years, the prevailing model explaining the gain-of- function activity of the R201 mutations was through the loss of GTPase activity and resulting inability of mutant G?s to switch off to the GDP state. Recently, our laboratory revised this model and revealed that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound G?s through stabilization of an intramolecular hydrogen bond network. This understanding has led to a therapeutic opportunity that we seek to exploit to treat pancreatic tumorigenesis. We propose to develop state-selective G?s binding molecules which block adenylyl cyclase (AC) activation. Inspired by the cyclic peptide natural product YM-254890 which is a GDP-state specific cyclic peptide inhibitor of G?q, we initiated a drug discovery approach to identify both active state and inactive state specific inhibitors of G?s. Using the Random non-standard Peptide Integrated Discovery (RaPID) system developed by our collaborator Dr. Hiroaki Suga we have selected active state and inactive state preferring cyclic peptides against G?s. We have solved high resolution X-ray co-crystal structures of our function blocking cyclic peptides which explain their nucleotide state specificity and inhibitory activity. We propose to use the RaPID technology to focus chemical diversity to improve potency of the lead cyclic peptides and test our lead molecules in cells generated from Dr. Bardeesy's model. This proposal capitalizes on three recent breakthroughs, mouse modeling by Dr. Bardeesy, unnatural cyclic peptide library generation by Dr. Suga, and a new non-canonical type of G protein signaling by the driver oncoprotein G?s by our laboratory to target a deadly form of pancreatic cancer.